Recently, there have been several reports demonstrating improvements in the flow cytometric detection of intracellular cytokines. These advances, although significant, have not yielded techniques that have easily been translated into broad use. To address this issue, we have coupled a fixation and permeabilization method with the use of directly labelled monoclonal anti-cytokine antibodies, providing both improved signal and simpler staining. The specificity of this technique can be demonstrated by blocking cytokine staining using a molar excess of recombinant cytokine. Additionally, unlabelled anti-cytokine antibodies block specific staining of labelled antibody, providing an objective means to place statistical markers. Using such controls, we routinely detect as few as 0.1% false positive cells, allowing the flow cytometric detection of IL-5. This technique was used to detect intracellular cytokines. We examined peripheral blood T cell subpopulations to determine the cytokines produced by individual lymphocytes. Cells from normal individuals and helminth infected patients were sorted for the CD4+CD27+ and CD4+CD27- subpopulations. Intracellular staining for IL-4, IL-5, and IFN-gamma revealed that virtually no CD4+CD27- lymphocytes produce both IL-5 and IFN-gamma while a distinct proportion produced both IL-4 and IFN-gamma (0.1-8.0%) and 66-84% of IL-5-producing cells also produce IL-4. Lymphocytes from normals and patients had the same functional T cell subsets, but the CD4+CD27- lymphocytes from patients had higher frequencies of cells producing IL-4 or IL-5. Normal subjects had higher frequencies of cells producing IFN-gamma. We are now applying this technique to the characterization of cells entering the airways after segmental challenge with antigen. Initial results suggest that the lymphocytes that initially enter the airways are identical to the circulating lymphocyte population in their cytokine profile.